ABSTRACT Type 2 diabetes has reached epidemic proportions and is a leading cause of coronary heart disease in the United States. Insulin resistance, a hallmark of type 2 diabetes, is associated with a 2 to 4 times higher risk of cardiac morbidity and mortality following acute myocardial infarction (MI). The goals of this project are to elucidate the molecular mechanisms linking insulin resistance to poor cardiac recovery after MI and to apply this knowledge to develop therapeutic strategies for improving recovery of cardiac function in diabetic patients at reperfusion. Uncoupling protein 3 (UCP3) is a mitochondrial anion carrier protein with antioxidant properties involved in the metabolism of long-chain fatty acids (LCFA). Muscle UCP3 content is 50% lower in type 2 diabetic patients compared with healthy control subjects. A similar decrease is observed in the heart of mice and rats with insulin resistance and type 2 diabetes. Using CRISPR/Cas9-targeted mutation in rats, we have gathered preliminary data showing that a 50% decrease in cardiac UCP3 levels is sufficient to significantly impair contractile recovery following ischemia. Our results further suggest that decreased functional recovery of insulin resistant and UCP3 deficient hearts after ischemia is caused by a limited capacity to oxidize LCFA at reperfusion, a defect that can be rescued by supplying medium-chain fatty acids (MCFA) as an alternative fuel. Besides the bioenergetic deficit, impaired LCFA oxidation is known to cause a toxic accumulation of long-chain ceramides and to increase oxidative stress. Therefore, we hypothesize that decreased UCP3 impairs the recovery of systolic function in insulin resistant hearts following MI by limiting myocardial LCFA oxidation, increasing mitochondrial dysfunction, and increasing cardiac myocyte death at reperfusion. Three aims will address this hypothesis in mouse and rat models of dietary, pharmacologically, or genetically induced myocardial insulin resistance or UCP3 deficiency (40-50% decrease) in a multisystem approach combining in vivo models of MI/reperfusion to isolated beating hearts to isolated mitochondria. Aim 1 will examine the effect of UCP3 deficiency and of its reversal on cardiac structural and functional recovery post MI/reperfusion. Aim 2 will investigate the molecular consequences of UCP3 deficiency for mitochondrial function and cardiac oxidative metabolism during ischemia/reperfusion. Aim 3 will test whether a metabolic intervention based on increasing supply of MCFA to the heart can reverse these abnormalities. We expect this project to reveal a novel molecular mechanism responsible for the poor prognosis of type 2 diabetic patients following MI/reperfusion, and that it will provide the basis for additional studies to test MCFA-based treatments as a metabolic strategy to improve cardiac outcomes in T2DM patients undergoing reperfusion after MI.